Purification, Characterization, and Preliminary X-Ray Diffraction Analysis of a Lactose-Specific Lectin from Cymbosema roseum Seeds |
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Authors: | Bruno A M Rocha Frederico B M B Moreno Plínio Delatorre Emmanuel P Souza Emmanuel S Marinho Raquel G Benevides Joane Kathelen Rodrigues Rustiguel Luis A G Souza Celso S Nagano Henri Debray Alexandre H Sampaio Walter F de Azevedo Jr Benildo S Cavada |
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Institution: | BioMol-Lab, Departmento de Bioquímica e Biologia Molecular, Universidade Federal do Ceará, P. O. Box 6043, 60.455-970, Fortaleza, Ceará, Brazil. |
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Abstract: | The unique carbohydrate-binding property of lectins makes them invaluable tools in biomedical research. Here, we report the purification, partial primary structure, carbohydrate affinity characterization, crystallization, and preliminary X-ray diffraction analysis of a lactose-specific lectin from Cymbosema roseum seeds (CRLII). Isolation and purification of CRLII was performed by a single step using a Sepharose-4B-lactose affinity chromatography column. The carbohydrate affinity characterization was carried using assays for hemagglutination activity and inhibition. CRLII showed hemagglutinating activity toward rabbit erythrocytes. O-glycoproteins from mucine mucopolysaccharides showed the most potent inhibition capacity at a minimum concentration of 1.2 microg mL(-1). Protein sequencing by mass spectrometry was obtained by the digestion of CRLII with trypsin, Glu-C, and AspN. CRLII partial protein sequence exhibits 46% similarity with the ConA-like alpha chain precursor. Suitable protein crystals were obtained with the hanging-drop vapor-diffusion method with 8% ethylene glycol, 0.1 M Tris-HCl pH 8.5, and 11% PEG 8,000. The monoclinic crystals belong to space group P2(1) with unit cell parameters a = 49.4, b = 89.6, and c = 100.8 A. |
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Keywords: | Cymbosema roseum Crystallization Tandem mass spectrometry Lectins Lactose-specific lectin |
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